Mitochondria play critical roles in both the life and death of cardiac myocytes. They are important generators of energy, providing ATP through oxidative phosphorylation. However, mitochondria also monitor complex information from the environment and intracellular milieu, including the presence or absence of growth factors, oxygen, reactive oxygen species, and DNA damage. Thus, it is not surprising that there is a strong link between mitochondrial dysfunction and cardiovascular disease. The Bcl-2 family proteins control mitochondrial outer membrane permeabilization and play a key role in regulating the mitochondrial apoptotic pathway. Mcl-1 is an anti-apoptotic Bcl-2 protein which is expressed at higher levels in the myocardium compared to other anti-apoptotic proteins such as Bcl-2 and Bcl-XL. Surprisingly, little is known about how Mcl-1 regulates cell survival in myocardial cells. We therefore generated a heart specific inducible knockout of Mcl-1 and discovered that loss of Mcl-1 in cardiac myocytes led to rapid mitochondrial dysfunction and cell death. Surprisingly, Mcl-1 deficient myocytes displayed signs of necrotic cell death instead of apoptotic cell death, suggesting that besides its anti-apoptotic role, Mcl-1 has an essential but yet unidentified role in maintaining mitochondrial function in cardiac myocytes. To better understand the physiological function(s) of Mcl-1 in the heart, we plan to identify new proteins that interact with Mcl-1 and elucidate the functional significance of this interaction in Aim 1. Mitochondria are highly dynamic organelles that are constantly undergoing fission and fusion, and these processes play important roles in the normal turnover of mitochondria. Defects in these processes can affect mitochondrial function and cell survival. We found that Mcl-1 can influence mitochondrial dynamics by inducing fission. Thus, in Aim 2, we will examine if Mcl-1 regulates mitochondrial dynamics by recruiting components of the mitochondrial fission machinery and whether this process is essential for normal bioenergetic function. Removal of dysfunctional mitochondria by autophagy is an essential process, and defects in this pathway in the heart lead to accumulation of dysfunctional mitochondria and development of heart failure. Ultrastructural analysis revealed a lack of mitochondrial autophagy in myocytes lacking Mcl-1, suggesting that Mcl-1 deficient myocytes are not delivering damaged mitochondria to autophagosomes. In Aim 3, we will investigate the role of Mcl-1 in regulating mitochondrial autophagy. Mitochondrial turnover decreases with age resulting in accumulation of dysfunctional mitochondria in the cell. Therefore, in Aim 4, we will investigate if enhanced levels of Mcl-1 will protect against cardiomyopathic challenge and as well as prolong survival of cardiac myocytes in aging using wild type and Mcl-1 transgenic mice. This project will provide important new insights into mitochondrial function in cardiac myocytes and how mitochondrial dysfunction contributes to development of cardiovascular disease. (End of Abstract)